1. Field of the Invention
The present invention discloses a method for expressing multiple recombinant proteins in the milk of transgenic non-human mammals, characterized in which human clotting factor IX gene and porcine lactoferrin gene are transferred into the mammal by gene injection and embryonic implantation to obtain expression in the milk of transgenic mammal and its offsprings.
2. Description of Related Art
In clinical medicine, there are many cases involving the inability of in-vivo synthesis of bioactive protein due to genetic defect. This kind of diseases is usually managed by direct replenishment with recombinant proteins. Human clotting factor IX (hFIX) is a single chain glycoprotein with a molecular weight of 55 kDa. This protein is synthesized in liver and secreted into blood stream after translational modification. The level of hFIX in normal human plasma is about 5 μg/ml. In the plasma of patients suffered from hemophilia B, hFIX is absent, hence causing abnormal clotting function. It is known that hFIX gene is located in the long arm of X chromosome. Thus hemophilia B is a sex-linked hereditary disease, prevalent in males with average incidence of 1/30,000 (Thompson, 1986). Presently hFIX used on hemophilia B patients come from purified human plasma which carries the risk of infection (e.g. hepatitis and AIDS). If hFIX gene and tissue-specific regulatory sequence, such as α-lactalbumin regulatory sequence, are to be constructed into a recombinant expression plasmid, and then applying gene transfer technology to produce transgenic animal, it is possible to extract large quantity of hFIX with bioactivity in the animal milk without the concern about viral infection. The technique of mass-producing hFIX in the mammary glands of transgenic animals will provide great benefit for hemophilia B patients.
Recombinant hFIX cDNA was first expressed in the liver of transgenic mouse (Choo et al., 1987). It has been demonstrated that transgenic mouse carrying recombinant hFIX through gene transfer could express the products of said recombinant gene in its liver, and the level of liver-secreted hFIX in its blood was seven folds the level in normal human plasma (Jallat et al., 1990). Those experiments also found that when the recombinant hFIX retains the intron sequence, the expression efficiency of transgene is markedly elevated. Clark et al. (1989) also constructed a recombinant gene containing β-lactoglobulin (BLG) promoter and hFIX cDNA with mammary-specific expression for gene transfer in sheep and found that the resulting transgenic sheep could express the bioactive products of hFIX gene, but the expression level was only 25 ng/ml. Van Cott et al. (1999) fused mouse whey acidic protein promoter with the structural gene of hFIX and then transferred the recombinant gene into porcine chromosome. The result showed that the expression of transgene in the mammary gland of transgenic sow reached as high as 0.2 mg/ml, but the hFIX gene expression stopped during lactation. Based on the study results described above, there is high commercial value in the R&D of mass producing recombinant hFIX using the mammary gland of transgenic animals. Still, there are several obstacles to be overcome. For example, the expression level of recombinant hFIX in the milk of transgenic animal, the inability to sustain expression until the end of lactation, and improving the health of swine after hFIX gene and other genes of additive effect are co-transferred into the swine. These are the goals current research endeavors are attempting to obtain.